From Paris Descartes University and Hôpital Necker, Assistance Publique-Hôpitaux de Paris, France (A.L., J.P.D.V.H., M.R.); Paris Translational Research Centre for Organ Transplantation, INSERM, UMR-S970, France (A.L., J.P.D.V.H., O.A., T.B., J.P.E., C.L., P.B., X.J.); Pathology Department, Necker Hospital, Paris, France (J.P.D.V.H.); Alberta Transplant Applied Genomics Centre; University of Alberta, Edmonton, AB, Canada (L.H., J.R., J.M.V., K.F., P.F.H.); Cardiology Department and Intensive Care (M.C.B.), Cardiology and Heart Transplant Department (R.G., X.J.), and Pathology Department (P.B.), Georges Pompidou Hospital, Paris, France; Pathology (P.R.) and Cardiac Surgery Departments (S.V., P.L.), La Pitié Salpétrière Hospital, Paris; Pathology (C.T.) and Thoracic and Cardiovascular Surgery Departments (S.P), Laennec Hospital, Nantes; Pathology (A.F.) and Cardiovascular Surgery Departments (A.G), Charles Nicolle Hospital, Rouen, France.
Circulation. 2017 Mar 7;135(10):917-935. doi: 10.1161/CIRCULATIONAHA.116.022907. Epub 2017 Feb 1.
Antibody-mediated rejection (AMR) contributes to heart allograft loss. However, an important knowledge gap remains in terms of the pathophysiology of AMR and how detection of immune activity, injury degree, and stage could be improved by intragraft gene expression profiling.
We prospectively monitored 617 heart transplant recipients referred from 4 French transplant centers (January 1, 2006-January 1, 2011) for AMR. We compared patients with AMR (n=55) with a matched control group of 55 patients without AMR. We characterized all patients using histopathology (ISHLT [International Society for Heart and Lung Transplantation] 2013 grades), immunostaining, and circulating anti-HLA donor-specific antibodies at the time of biopsy, together with systematic gene expression assessments of the allograft tissue, using microarrays. Effector cells were evaluated with in vitro human cell cultures. We studied a validation cohort of 98 heart recipients transplanted in Edmonton, AB, Canada, including 27 cases of AMR and 71 controls.
A total of 240 heart transplant endomyocardial biopsies were assessed. AMR showed a distinct pattern of injury characterized by endothelial activation with microcirculatory inflammation by monocytes/macrophages and natural killer (NK) cells. We also observed selective changes in endothelial/angiogenesis and NK cell transcripts, including CD16A signaling and interferon-γ-inducible genes. The AMR-selective gene sets accurately discriminated patients with AMR from those without and included NK transcripts (area under the curve=0.87), endothelial activation transcripts (area under the curve=0.80), macrophage transcripts (area under the curve=0.86), and interferon-γ transcripts (area under the curve=0.84; <0.0001 for all comparisons). These 4 gene sets showed increased expression with increasing pathological AMR (pAMR) International Society for Heart and Lung Transplantation grade (<0.001) and association with donor-specific antibody levels. The unsupervised principal components analysis demonstrated a high proportion of molecularly inactive pAMR1(I+), and there was significant molecular overlap between pAMR1(H) and full-blown pAMR2/3 cases. Endothelial activation transcripts, interferon-γ, and NK transcripts showed association with chronic allograft vasculopathy. The molecular architecture and selective AMR transcripts, together with gene set discrimination capacity for AMR identified in the discovery set, were reproduced in the validation cohort.
Tissue-based measurements of specific pathogenesis-based transcripts reflecting NK burden, endothelial activation, macrophage burden, and interferon-γ effects accurately classify AMR and correlate with degree of injury and disease activity. This study illustrates the clinical potential of a tissue-based analysis of gene transcripts to refine diagnosis of heart transplant rejection.
抗体介导的排斥反应（AMR）是导致心脏移植物丧失的原因之一。然而，AMR 的病理生理学以及如何通过移植组织内基因表达谱来改善免疫活性、损伤程度和分期的检测，仍然存在重要的知识空白。
我们前瞻性地监测了来自法国 4 个移植中心的 617 名心脏移植受者（2006 年 1 月 1 日至 2011 年 1 月 1 日）的 AMR。我们将 55 名 AMR 患者与 55 名无 AMR 的匹配对照组进行比较。我们使用组织病理学（国际心肺移植协会 2013 级）、免疫染色和活检时的循环抗 HLA 供体特异性抗体，以及使用微阵列对移植组织进行系统的基因表达评估，对所有患者进行了特征描述。效应细胞通过体外人细胞培养进行评估。我们研究了在加拿大埃德蒙顿接受心脏移植的 98 名患者的验证队列，其中包括 27 例 AMR 和 71 例对照。
共评估了 240 例心脏移植心内膜心肌活检。AMR 表现出独特的损伤模式，其特征为内皮细胞激活伴单核细胞/巨噬细胞和自然杀伤（NK）细胞的微循环炎症。我们还观察到内皮/血管生成和 NK 细胞转录物的选择性变化，包括 CD16A 信号和干扰素-γ诱导基因。AMR 选择性基因集能够准确地区分 AMR 患者和无 AMR 患者，包括 NK 转录物（曲线下面积=0.87）、内皮激活转录物（曲线下面积=0.80）、巨噬细胞转录物（曲线下面积=0.86）和干扰素-γ转录物（曲线下面积=0.84；所有比较均<0.0001）。这些 4 个基因集的表达随着国际心肺移植协会（ISHLT）组织学 AMR 分级（pAMR）的增加而增加（p<0.001），并与供体特异性抗体水平相关。无监督主成分分析显示，分子上无活性的 pAMR1（I+）比例较高，pAMR1（H）和完全 pAMR2/3 病例之间存在显著的分子重叠。内皮激活转录物、干扰素-γ和 NK 转录物与慢性移植物血管病有关。在发现组中确定的基于组织的特定发病机制转录物的分子结构和选择性 AMR 转录物，以及基因集鉴别能力，在验证组中得到了重现。
反映 NK 负荷、内皮激活、巨噬细胞负荷和干扰素-γ效应的基于组织的特定发病机制转录物的测量可以准确地对 AMR 进行分类，并与损伤程度和疾病活动相关。本研究说明了基于组织的基因转录分析在细化心脏移植排斥诊断方面的临床潜力。
